1. Field of the Invention
The present invention relates to a method for the production of hybrid polynucleotide molecules from two parental polynucleotide molecules using single-stranded polynucleotides, which can achieve superior effects to the existing methods, such as a higher frequencies.
2. Description of the Related Art
DNA shuffling is a developed technique that allows accelerated and directed protein evolution in vitro. In this method, the acquisition of genes encoding improved proteins is done in two steps. In the first step, a single gene is mutagenized, and desired mutant genes are selected. In the second step, the mutant genes are fragmented by DNase I, and subsequently recombined in vitro by using PCR. Among the recombinants, (i.e., the products of DNA shuffling), those producing most favored proteins are isolated (FIG. 1; 1, 2). The modified versions of the DNA shuffling exist: (i) random priming was used to generate DNA fragment instead of the DNase I digestion (3); (ii) PCR conditions with very short annealing/extension steps were employed to increase the frequency of recombination (4). Using these techniques, a number of improved enzymes have been obtained (1-9). When the DNA shuffling is done using a set of homologous genes instead of a set of mutant genes derived from a single gene, this technique is called family shuffling. Family shuffling utilizes naturally occurring nucleotide substitutions among family genes as the driving force for the in vitro evolution. The application of the family shuffling strategy has also provided many successful examples (10-15).
A potential problem of the family shuffling is a low yield of recombinants (i.e., hybrid molecules constituted from several family gene sequences). When two parental genes of 80% nucleotide sequence identity were shuffled, the frequency of hybrid formation was less than 1% (16-17). The low recombination yield may be due to a lower frequency of the heteroduplex formation compared to the frequency of the homoduplex formation (FIG. 2; 16).
Thus, it is desirable to develop methods which allow for the high frequency production of recombinant molecules of two or more family genes. We have reported a technique which is the shuffling of restriction endonuclease-digested DNA fragments instead of the shuffling of randomly fragmented DNA (16). The annealing of endonuclease-digested DNA fragments would produce homoduplex at a high frequency, but significant DNA elongation only occurs on the heteroduplex molecules.